Targeted Gene Therapies for Pain and Other Neuro-Related Disorders

ABSTRACT

Provided herein are nucleic acids for expressing modified ligand-gated ion channel proteins in excitable cells or secretory cells, such as nerves and neurons and optionally including viral sequences, such as Adeno-associated virus sequences, for delivery to excitable cells or secretory cells of a patient. Also provided herein are methods of modulating cell membrane potentials in an excitable cell or secretory cell, and for treatment of a disease or disorder associated with the nervous system in a patient, such as chronic pain or itch.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/493,387, filed Mar. 20, 2018, which is the United Statesnational phase of International Application No. PCT/US2018/023364 filedMar. 20, 2018, and claims the benefit of U.S. Provisional PatentApplication No. 62/473,630 filed Mar. 20, 2017, each of which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via Patent Center and is hereby incorporated byreference into the specification in its entirety. The name of the filecontaining the Sequence Listing is 2304751.xml. The size of the file is73,585 bytes, and the file was created on Jun. 1, 2023.

Provided herein are compositions and related methods useful fortreatment of pain, especially chronic pain.

Over 100 million people in the U.S. suffer from debilitating pain causedby disease and trauma. Current therapies are focused on opioids andNSAIDs, but these treatments lose efficacy or produce serious sideeffects. Addiction to opioid-based prescription painkillers has led toan epidemic level rise in opioid-related overdose deaths. Because of thelack of adequate treatment options, pain continues to be a majorclinical problem. Therapeutic solutions to the lack of adequate paintreatment, especially for chronic pain, are needed.

SUMMARY

Provided herein are nucleic acids for tissue-specific delivery ofmodified ligand-gated ion channels (LGICs) that can be selectivelyactivated with tailored compound ligands. Such LGICs, once delivered tothe neurons of interest by gene therapy methods, would render thoseneurons sensitive to a ligand selective for such novel LGICs and wouldobviate the need for local delivery of the ligand, since the tailoredligand would have no effect on native LGICs. Furthermore, selectiveactivation of those tissue-targeted LGICs would eliminate thenon-specific effects arising from activation of neighboring populationsof neurons that inevitably occur due to the ubiquitous expression ofnative LGICs. This provides specificity for control of neuron activitythat can be used therapeutically to treat neurological diseases andconditions, such as chronic pain or itch. Therefore, development ofnovel tissue-targeted LGICs with unique pharmacology has therapeuticutility. Related methods also are provided.

According to a first aspect of the invention, a nucleic acid isprovided, comprising a gene for expressing a modified ligand-gated ionchannel. The gene comprises an open reading frame encoding a modifiedligand-gated ion channel under transcriptional control oftranscriptional control elements governing cell-specific expression inCNS neurons, such as dorsal horn neurons, spinal cord cells, or braincells, or in inhibitory neurons or nerve cells. Examples oftranscription control elements include: a CCK promoter, a Tac1 promoter,an NTS promoter, an NMU promoter, a Calb1 promoter, an SST promoter, aGRPR promoter, a parvalbumin promoter, a Gal promoter, an NPY promoter,a PKCγ promoter, or a Calb2 promoter. The modified ligand-gated ionchannel comprises a modified ligand binding domain activatable by anexogenous ligand, and optionally selective to the exogenous ligand, andan ion pore domain.

In another aspect of the invention, a method is provided of modulating(increasing or decreasing) the membrane potential of an excitable cellor a secretory cell. The method comprises expressing in the cell agenetic construct comprising a gene for expressing a modifiedligand-gated ion channel, comprising an open reading frame encoding amodified ligand-gated ion channel under transcriptional control oftranscriptional control elements governing cell-specific expression inCNS neurons, such as dorsal horn neurons, spinal cord cells, or braincells, or in inhibitory neurons or nerve cells, such as a CCK promoter,a Tac1 promoter, an NTS promoter, an NMU promoter, a Calb1 promoter, anSST promoter, a GRPR promoter, a parvalbumin promoter, a Gal promoter,an NPY promoter, a PKCγ promoter, or Calb2 promoter and a modifiedligand-gated ion channel comprising a modified ligand binding domainactivatable by an exogenous ligand, and optionally selective to theexogenous ligand, and an ion pore domain, and contacting the cell withan amount of the exogenous ligand effective to activate the modifiedligand gated ion channel thereby modulating the membrane potential ofthe cell.

In a further aspect of the invention, a method is provided of treating adisease or disorder associated with the nervous system in a patient. Themethod comprises delivering a nucleic acid as described below, andadministering the exogenous ligand to the patient in an amount effectiveto activate a modified ligand gated ion channel in a patient therebytreating the disease or disorder associated with the nervous system inthe patient. The nucleic acid comprises a gene for expressing themodified ligand-gated ion channel. The gene comprises an open readingframe encoding the modified ligand-gated ion channel undertranscriptional control of transcriptional control elements governingcell-specific expression in CNS neurons, such as dorsal horn neurons,spinal cord cells, or brain cells, or in inhibitory neurons or nervecells. Examples of transcription control elements include: a CCKpromoter, a Tac1 promoter, an NTS promoter, an NMU promoter, a Calb1promoter, an SST promoter, a GRPR promoter, a parvalbumin promoter, aGal promoter, an NPY promoter, a PKCγ promoter, or a Calb2 promoter. Themodified ligand-gated ion channel comprises a modified ligand bindingdomain activatable by an exogenous ligand, and optionally selective tothe exogenous ligand, and an ion pore domain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1 depict exemplary nucleic acid sequences, continuous overFIGS. 1A-10 , comprising promoter sequences useful in the geneticconstructs described herein, as follows: Human CCK Promoter (SEQ ID NO:1), Human Tac1 Promoter (SEQ ID NO: 2), Human Nts Promoter (SEQ ID NO:3), Human Nmu Promoter (SEQ ID NO: 4), Human Calb1 Promoter (SEQ ID NO:5), Human Parvalbumin Promoter (SEQ ID NO: 6), Human Gal Promoter (SEQID NO: 7), Human NPY Promoter (SEQ ID NO: 8), Human SST Promoter (SEQ IDNO: 9), Human GRPR Promoter (SEQ ID NO: 10), Human PRKCG Promoter (SEQID NO: 11), Human Calb2 Promoter (SEQ ID NO: 12), Mouse CCK Promoter(SEQ ID NO: 13), Mouse Calb2 Promoter (SEQ ID NO: 14), Mouse PRKCGPromoter (SEQ ID NO: 15), Mouse Calb1 Promoter (SEQ ID NO: 16), andMouse Nmu Promoter (SEQ ID NO: 17).

FIG. 2 provides amino acid sequences of exemplary a7-nicotinicacetylcholine receptor ligand binding domains (SEQ ID NOs: 18-20 asindicated in the figure).

FIGS. 3A-3E provide exemplary amino acid sequences for chimeric LGICs.FIG. 3A provides an exemplary amino acid sequence of an α7-5HT3 chimericreceptor (SEQ ID NO: 21), including a human α7 nAChR LBD and a murine5HT3 IPD components. FIG. 3B provides an exemplary amino acid sequenceof α7-GIyR chimeric receptor (SEQ ID NO: 22) including a human α7 nAChRLBD) and a human GIyR IPD components. FIG. 3C provides an exemplaryamino acid sequence of α7-5HT3 chimeric receptor (SEQ ID NO: 23,including human α7 nAChR LBD and a human 5HT3 IPD components. FIG. 3Dprovides an exemplary amino acid sequence of α7-GABAC chimeric receptor(SEQ ID NO: 24), including a human α7 nAChR LBD and a human GABAC IPDcomponents. FIG. 3E provides an exemplary amino acid sequence of ratnAChR sequence (SEQ ID NO: 25).

FIG. 4 provides exemplary exogenous ligands (U.S. 2018/0009862 A1).

FIG. 5 . (a) Cre-dependent construct for expressing PSAM-GIyR in neuronsusing AAV delivery. PSEM^(89S) activates the PSAM-GIyR channel,preventing action potential firing of the cells expressing PSAM-GIyR.(b) Dorsal horns of CCK^(Cre) and Calr^(Cre) mice injected intraspinallywith AAV8-Flex-PSAM-GIyR virus shows binding ofalpha-bungarotoxin-Alexa647 ((α-BTX-Alexa647) to PSAM-GIyR in patternsconsistent with the distribution of these two genes.

FIGS. 6A and 6B. Positive and negative controls for PSAM-GIyR andPSEM^(89S). (a) Schematic of PSAM-GIyR construct with GFP reporter usedto make AAV8-hSyn-Flex-PSAM-GIyR. Virus was injected into Tlx3^(Cre)mice for electrophysiological slice recordings. (b) Electrode impaling aGFP+ cell (also shown as DI, image) in a transverse slice of the dorsalhorn from a Tlx3^(Cre) mouse injected with AAV8-hSyn-Flex-PSAMGIyRvirus. (c) Bath application of 30 μM PSEM^(89S) induces a sustainedhyperpolarization specifically in GFP-expressing cells. (d) Currentinjected into GFP+ cell produces action potentials (Pre) that arecompletely blocked after application of PSEM^(89S) (30 μM) to the slice.(e) Control for the specificity of α-BTX-Alexa647. Contralateral dorsalhorn lacks staining for α-BTX-Alexa647. Calretinin; PKCγ. Scale bar=50μm. Box indicates area of inset. (f) In mice lacking PSAM-GIyR,mechanical allodynia and heat hypersensitivity induced by thecarrageenan pain model are not altered by injection of PSEM^(89S) (30mg/kg, i.p.) (p>0.999, n=4) (g) Punctate and dynamic allodynia inducedby CFA are also not altered by injection of PSEM^(89S) (p>0.999, n=6).(h) In the mice lacking PSAM-GIyR, punctate and dynamic allodyniainduced by the sural-SNI model are not altered by injection ofPSEM^(89S) (30 mg/kg, i.p.) (n>0.999, n=6). Data are mean±standard errorof the mean (SEM). ***P<0.001; ns=not significant (i.e. p>0.05)

FIG. 7 . CCK⁺ excitatory neurons in the dorsal horn primarily laminaIII-IV are not required for baseline somatosensory behavior or motorbehavior on rotarod. (a) Injection of PSEM^(89S) into CCK^(Cre) miceexpressing PSAM-GIyR ipsilaterally in the dorsal horn has no effect onbaseline von Frey threshold (p=0.2192, n=8), response to cotton swab(p=0.7293, n=8), pressure with calibrated calipers (p=0.7450, n=8) orpinprick (p=0.5983, n=8). Hargreaves latencies are unchanged (p=0.9746,n=8). (b) Performance on the rotarod is also not altered by injection ofPSEM^(89S). Data are mean±SEM. None are significantly different.

FIG. 8 . CCK+ excitatory dorsal horn neurons are required for thetransmission of persistent forms of pain. Mice expressing PSAM-GIyR inCCK+ dorsal horn neurons were tested in persistent pain behaviors. (a)In the carrageenan pain model, injection of PSEM^(89S) (30 mg/kg, i.p.)reversed punctate (p=0.0017, n=8) and dynamic allodynia (p=0.0001, n=8)as well as heat hypersensitivity (p=0.0002, n=8). (b) In the CFA model,PSEM^(89S) injection also markedly reversed punctate (p=0.0034, n=5) anddynamic allodynia (p=0.0016, n=5) as well as heat hypersensitivity(p=0.0368, n=5). (c,d) Inhibiting the CCK population reversed punctateallodynia in the sural-SNI (p=0.0003, n=12) and tibial-SNI (p=0.0054,n=5) models. Dynamic allodynia in sural-SNI is also reversed (p=0.0001,n=12). (e,f) PSEM^(89S) also reversed heat hypersensitivity induced byMG (p=0.0272, n=4) or multi-dose STZ (p=0.010, n=9). Data are mean±SEM.*p<0.05, **p<0.01, ***P<0.001.

FIG. 9 . Calretinin⁺ excitatory neurons in the dorsal horn lamina II arenot required for baseline somatosensory behavior or motor behavior onthe rotarod. (a) Injection of PSEM^(89S) (30 mg/kg, i.p.) intoCalr^(Cre) mice expressing PSAM-GIyR ipsilaterally in the dorsal hornhas no effect on ipsilateral von Frey threshold (p=0.3988, n=8),response to cotton swab (p=0.7963, n=8), pressure with calibratedcalipers (p=0.8350, n=8) sticky tape (p=0.9346, n=8) or pinprick(p=0.7689, n=8). Hargreaves latencies are also unchanged (p=0.6125,n=8). (b) Motor behavior measured by rotarod is not altered byinhibition of dorsal horn calretinin neurons with PSEM^(89S). Data aremean±SEM. None are significantly different.

FIG. 10 . Calretinin⁺ excitatory neurons in lamina II of the dorsal hornconvey mechanical allodynia induced by inflammatory, but not neuropathicpain models. Inhibition of PSAM-GlyR expressing calretinin neurons inthe dorsal horn lamina II by injection of PSEM^(89S) (30 mg/kg, i.p.)reverses mechanical allodynia induced by models of inflammatory painincluding (a) carrageenan (p=0.0074, n=8), (b) CFA (p=0.0026, n=8), (c)incision (punctate, p=0.0028, n=7; dynamic p=0.0001, n=7) Data aremean±SEM. **p<0.01, ***P<0.001.

FIG. 11 : AAV-mCCK-PSAM4-WPRE construct. A 2.5 kb fragment of the mouseCCK promoter was sub-cloned upstream of the PSAM (L131G Q139LY217F)-GlyR (PSAM4) gene with a chimeric intron (135 bp) located inbetween the promoter and the PSAM4 gene.

FIG. 12 . Expression of PSAM4 in the dorsal horn of an adult wildtypeC57BI/6 mouse. α-bungarotoxin-Alexa647 (α-BTXAlexa-647) staining of thelumbar dorsal horn of a mouse unilaterally injected with the AAV8mCCK-PSAM4-WPRE virus in the dorsal horn (1¹³ vg/ml, custom packaged).

FIG. 13 . Varenicline has no effect on baseline mechanical and heatsensitivity or CFA or sural SNI induced hypersensitivity in mice lackingPSAM4. PWT, frequency of response and PWL were tested in adult wildtypeC57BI/6 mice (not expressing PSAM4) before and after injection ofvarenicline (0.1 mpk, i.p) at baseline and 3 and 7 days after injectionof CFA or sural-SNI surgery, respectively. Data are mean±SEM. N=5 mice.*p<0.05, **p<0.01, ***P<0.001; ns=not significant (i.e. p>0.05).

FIG. 14 . Control showing effect of varenicline on mechanical and heatsensitivity at baseline in mice injected with mCCK-PSAM4 virus. Theeffect of varenicline (0.3 mpk, i.p) on baseline mechanical and heathypersensitivity was tested 2 weeks after injection of AAV8 mCCK-PSAM4unilaterally into the dorsal horn. Injection of varenicline had noeffect on baseline PWT, percentage response or PWL (measured 30-60minutes following injection of the drug) in mice expressing PSAM4 indorsal horn CCK+ neurons. Data are mean±SEM. N=5-6 mice. **p<0.05,**p<0.01, ***P<0.001; ns=not significant (i.e. p>0.05).

FIG. 15 . Varenicline reverses mechanical allodynia induced by CFA inmice expressing mCCK-PSAM4 in the dorsal horn. Adult wildtype C57BI/6mice injected with AAV8 mCCK-PSAM4-WPRE virus unilaterally in the dorsalhorn. PWT to von Frey filaments and percentage of response to cottonswab were measured before and 3 days after CFA injection in plantarhindpaw and after injection of varenicline (0.3 mpk, i.p). Injection ofvarenicline reversed both PWT and response frequency to cotton swabafter CFA. Data are mean±SEM. N=5 mice. *p<0.05, **p<0.01, ***P<0.001.

FIG. 16 . Varenicline reverses mechanical allodynia induced by the suralmodel of SNI in mice expressing mCCK-PSAM4 in the dorsal horn. Adultwildtype C57BI/6 mice injected with mCCK-PSAM4 unilaterally in thedorsal horn. Three weeks later, PWT to von Frey filaments was measuredbefore and 7 days after sural-SNI surgery on the ipsilateral side andthen again after injection of varenicline (0.3 mpk, i.p). Injection ofvarenicline reversed PWT after SNI. Data are mean±SEM. N=5 mice.**p<0.01, ***p<0.001.

DETAILED DESCRIPTION

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges are both preceded by the word “about”. In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, unless indicated otherwise, the disclosure of ranges is intendedas a continuous range including every value between the minimum andmaximum values. As used herein “a” and “an” refer to one or more.

As used herein, the term “comprising” is open-ended and may besynonymous with “including”, “containing”, or “characterized by”. Theterm “consisting essentially of” limits the scope of a claim to thespecified materials or steps and those that do not materially affect thebasic and novel characteristic(s) of the claimed invention. The term“consisting of” excludes any element, step, or ingredient not specifiedin the claim. As used herein, embodiments “comprising” one or morestated elements or steps also include, but are not limited toembodiments “consisting essentially of” and “consisting of” these statedelements or steps.

A “patient” is a human or animal, e.g., vertebrates or mammals,including rat, mouse, rabbit, pig, monkey, chimpanzee, cat, dog, horse,goat, guinea pig, and birds, and does not imply or require adoctor-patient or veterinarian-patient relationship.

The terms “transfect”, “transfection”, “transfected”, and like termsrefer to the introduction of a gene into a eukaryotic cell, such as akeratinocyte, and includes “transduction,” which is viral-mediated genetransfer, for example, by use of recombinant AAV, adenovirus (Ad),retrovirus (e.g., lentivirus), or any other applicable viral-mediatedgene transfer platform.

By “expression” or “gene expression,” it is meant the overall flow ofinformation from a gene. A “gene” is a functional genetic unit forproducing a gene product, such as RNA or a protein in a cell, or otherexpression system encoded on a nucleic acid and generally comprising: atranscriptional control sequence, such as a promoter and othercis-acting elements, such as transcriptional response elements (TREs)and/or enhancers; an expressed sequence that typically encodes a protein(referred to as an open-reading frame or ORF) or functional/structuralRNA; and a polyadenylation sequence). A gene produces a gene product(typically a protein, optionally post-translationally modified or afunctional/structural RNA) when transcribed. By “expression of genesunder transcriptional control of,” or alternately “subject to controlby,” a designated sequence such as a promotor, it is meant geneexpression from a gene containing the designated sequence operablylinked (functionally attached, typically in cis) to the gene. A genethat is “under transcriptional control” of a promotor or transcriptioncontrol element, is a gene that is transcribed at detectably differentlevels in the presence of a transcription factor, e.g., in specificcells, as further described below, and in the context of the presentdisclosure, produces a difference in transcription levels when expressedin a specific cell type (e.g., where the promoter is a CCK promoter, thegene is preferentially expressed in cells that express CCK natively. A“gene for expression of” a stated gene product is a gene capable ofexpressing that stated gene product when placed in a suitableenvironment, that is, for example, when transformed, transfected,transduced, etc. into a cell, and subjected to suitable conditions forexpression. In the case of a constitutive promoter “suitable conditions”means that the gene typically need only be introduced into a host cell.In the case of an inducible promoter, such as the tissue specificpromoters described herein, “suitable conditions” means when factorsthat regulate transcription, such as DNA-binding proteins, are presentor absent, for example, an amount of the respective inducer is availableto the expression system (e.g., cell), or factors causing suppression ofa gene are unavailable or displaced—effective to cause expression of thegene.

Transcriptional control elements include promoters, enhancers,transcription factor-responsive elements (TREs, e.g., transcriptionfactor binding sequences), suppressors, introns, etc., as arebroadly-known. Additional transcription control elements, such as a WPRE(woodchuck hepatitis virus post-transcriptional regulatory element), oran intron, e.g., as shown below, which can increase expression fromcertain viral vectors, can be included in the gene.

Exemplary tissue specific promoters, specific to excitable cells orsecretory cells, e.g. of the central nervous system (CNS), such as,without limitation, neurons, sensory neurons, dorsal horn cells, spinalcord cells, brain cells, and inhibitory neurons, include, for exampleand without limitation, those promoter sequences depicted in FIGS.1A-1L, and described in Table 1.

TABLE 1 Location for Inhibitory or Physiological Promoter Expressionexcitatory LGIC Effect Cholecystokinin Dorsal horn inhibitory Blockspersistent (CCK) neuron pain Tachykinin (Tac1) Dorsal horn inhibitoryBlocks persistent neuron pain PKCγ (PRKCG) Dorsal horn inhibitory Blockspersistent neuron pain Neurotensin (NTS) Dorsal horn inhibitory Blockspersistent neuron pain Neuromedin U Dorsal horn inhibitory Blockspersistent (NMU) neuron pain Calbindin 1 (Calb1) Dorsal horn inhibitoryBlocks persistent neuron pain Calbindin 2 (Calb2) Dorsal horn inhibitoryBlocks persistent neuron pain Somatostatin (SST) Dorsal horn inhibitoryBlocks persistent neuron pain Gastrin related Dorsal horn inhibitoryBlocks itch peptide receptor neuron (GRPR) Galanin (GAL) Dorsal hornExcitatory Blocks persistent neuron pain or itch Neuropeptide Y Dorsalhorn Excitatory Blocks persistent (NPY) neuron pain or itch Parvalbumin(PV) Dorsal horn Excitatory Blocks persistent neuron pain or itch

A promoter is “specific” to specified excitable cells or secretory cellsif it causes gene expression in those cells of a gene to a sufficientextent for production of useful or therapeutically effective amounts ofthe described modified LGICs described herein in the specified excitablecells or secretory cells, and insignificant expression elsewhere in thecontext of the use, e.g. therapeutic use.

Although these are human sequences and consensus sequences, there isconservation among species and many promoter sequences that function inhuman cells will also be expected to do so in mice, or any mammal orvertebrate, and many promoter sequences that function in mice, or anymammal or vertebrate will also be expected to do so in human cells. Thesequences also may be modified, e.g., shortened, for virion packagingpurposes and optimal expression, so long as tissue-specificity of theconstruct remains.

One of the advantages of using highly circumscribed cell-type specificpromoters described herein, for example, Calb2 or CCK to driveexpression of PSAM4 or another LGIC (e.g., an inhibitory LGIC in Calb2or CCK expressing dorsal horn neurons), is that baseline mechanical orheat sensitivity are not affected (see, Examples 1 and 2, below) withexogenous ligand-mediated activation of the LGIC. These promotersprovide an important advantage over pan neuronal or pan excitatoryneuron or pan inhibitory neuron promoters, or also primary afferentpromoters such as TRPV1 (those neurons fibers are required for all heatsensation), which will negatively impact acute pain or touch in the areainnervated by the targeted neurons with exogenous ligand-mediatedactivation of the LGIC. The ability to feel acute pain is important toprotect the patient from bodily harm.

Production of useful nucleic acid constructs, such as recombinant viralvectors for production of nucleic acids, such as the genetic constructsand recombinant viral genomes described herein, is routine, in thatmolecular cloning and gene assembly methods are routine. Further, anumber of companies can custom-synthesize and verify multi-kilobasegenes, making the production of genes or genomes as described herein,such as rAAV or scAAV genomes, routine (See, e.g., Gene SynthesisHandbook, 2d Edition, 2014, GenScript USA, Inc.).

AAV (adeno-associated virus), is a virus belonging to the genusDependoparvovirus, and family Parvoviridae. The virus is a smallreplication-defective, non-enveloped virus. AAV is not currently knownto cause any disease by itself. AAV requires a helper virus, such asadenovirus or herpes simplex virus, to facilitate productive infectionand replication. In the absence of helper virus, AAVs establish a latentinfection within the cell, either by site-specific integration into thehost genome or by persisting in episomal forms. Gene therapy vectorsusing AAV can infect both dividing and quiescent cells. Furthermore, AAVserotypes have different tropism and can infect cells of multiplediverse tissue types. While eleven serotypes of AAV have been identifiedto date, AAV2 was among the first to be identified and has beenconsistently used for the generation of recombinant AAV vectors. Furthercertain natural or modified AAVs transduce specific organs or cellpopulations. In one example, AAV-PHP.eB and AAV-PHP.S, capsidsefficiently transduce the central and peripheral nervous systems,respectively, when administered intravenously (Chan, K. Y., et al.Engineered AAVs for efficient noninvasive gene delivery to the centraland peripheral nervous systems (2017) Nat. Neurosci 20(8):1172-1179).For example, compared to AAV9, AAV-PHP.B delivers genes to the brain andspinal cord at least 40 times more efficiently. See also, Tervo, D G, etal. A Designer AAV Variant Permits Efficient Retrograde Access toProjection Neurons (2016) Neuron 92, 372-382, describing engineered AAVvariants, e.g., rAAV2-retro, which permit robust retrograde access toprojection neurons with efficiency comparable to classical syntheticretrograde tracers, and enable sufficient sensor/effector expression forfunctional circuit interrogation and in vivo genome editing in targetedneuronal populations.

The AAV virion shell is approximately 25 nm in diameter and encapsulatesa single-stranded DNA genome that consists of two large open readingframes (ORFs) flanked by inverted terminal repeats (ITR). The ITRs arethe only cis-acting elements required for genome replication andpackaging. In wild-type AAV, the left ORF encodes four replicationproteins responsible for site-specific integration, nicking, andhelicase activity, as well as regulation of promoters within the AAVgenome. AAV possesses a 4.7 kb genome, and as such, efficient packagingof recombinant AAV (rAAV) vectors can be performed with constructsranging from 4.1 kb to 4.9 kb in size (see, e.g., Samulski, R J, et al.,AAV-Mediated Gene Therapy for Research and Therapeutic Purposes, Annu.Rev. Virol. 2014. 1:427-51).

Helper-free production of the rAAV requires transfection of thefollowing components into host cells, typically 293 cells (HEK293cells), which are broadly available, or similar cell lines: (1) an rAAVvector containing the transgene expression cassette flanked by the twoITRs; (2) expression of Rep and Cap proteins, typically provided by ahelper plasmid in trans; and (3) adenovirus genes encoding E1, E2A, E4,and virus-associated RNA, also provided, at least in part by anotherhelper plasmid in trans (293 cells produce the Ad E1 gene in trans). Repand Cap proteins, which are necessary for viral packaging, arereplication proteins and capsid proteins, respectively. Rep proteinsconsist of rep 78, 68, 52 and 40. They specifically are involved withthe replication of AAV. Cap proteins are comprised of three proteins,VP1, VP2 and VP3, with molecular weight of 87, 72 and 62 kDa,respectively. These capsid proteins assemble into a near-sphericalprotein shell of 60 subunits. Helper-free AAV packaging systems arebroadly available, for example, from Clontech of Mountain View,California, from Cell Biolabs, Inc. of San Diego, CA, and see, e.g.,U.S. Pat. Nos. 6,093,570, 6,458,587, 6,951,758, and 7,439,065. In scAAV(self-complementary AAV), the right ITR contains a deletion ofD-sequence (the packaging signal) and a terminal resolution sitemutation (Atrs), which prevent Rep-mediated nicking and force packagingof dimer or self-complementary genomes (see FIG. 8 ). Making dsAAV fromscAAV vector renders much improved transduction both in vitro and invivo (see, e.g., pscAAV-MCS Expression vector, Product Data Sheet, CellBiolabs, Inc., San Diego, California (2012-2016)).

Preparation of rAAV transducing particles, such as scAAV transducingparticles is routine. Since the transfection method is often consideredunsuitable for large-scale production, the infection of cell linesstably expressing Rep and Cap with adenovirus carrying a vector genomehas afforded the ability to scale-up. Another option includes infectionof proviral cell lines with adenovirus or herpes simplex virus vectorcarrying an AAV Rep and Cap expression cassette. These methods stillrequire the complete elimination of adenovirus (or herpesvirus) duringthe production process. However, in baculovirus expression vectorsystems for rAAV vector production in insect SF9 cells, the componentsof AAV production, including Rep and Cap proteins, as well as vectorgenomes are provided by separate recombinant baculoviruses. Ayuso, E.,“Manufacturing of recombinant adeno-associated viral vectors: newtechnologies are welcome”, Molecular Therapy—Methods & ClinicalDevelopment (2016) 3, 15049; doi:10.1038/mtm.2015.49, and Merten, O-W,et al., describe numerous robust current rAAV production methods, thoughcommercial scale-up and validation needs improvement. High viral titers(˜10¹²˜10¹³ vp/mL) may be required for certain uses described herein.Protocols are available in the literature for concentration andpurification of AAV vectors, allowing production of virus at these highconcentrations (see, e.g., Gray S J, et al. (2011) Production ofrecombinant adeno-associated viral vectors and use in in vitro and invivo administration. Curr Protoc Neurosci.doi:10.1002/0471142301.ns0417s57 and Guo P, et al. (2012) Rapid andsimplified purification of recombinant adeno-associated virus. J VirolMethods 183(2):139-146).

Once the virus has been produced in the, e.g., 293 cells, the cells arecollected, lysed, and the resultant virus is purified. Density gradientultracentrifugation, e.g., in cesium chloride or nonionic iodixanol(VISIPAQ™) gradients and column chromatography, such as ion-exchange,heparin-affinity, or mucin-affinity column chromatography, depending onthe AAV serotype. Once the rAAV has been purified and concentrated to asuitable concentration, the virus can be used for in vitro celltransduction or for in vivo animal injection at an appropriate MOI(Multiplicity of Infection).

Numerous rAAV vectors have been made containing genes for expressingfluorescent proteins, and are commercially available. A “gene” is agenetic element for production of a gene product such as a protein orRNA. A gene for production of a protein product includes, from 5′ to 3′according to convention: one or more regulatory elements (transcriptioncontrol elements) such as promoters, transcription response elements(TREs), repressors, enhancers; an open-reading frame (ORF) encoding aprotein or a sequence encoding a functional RNA; and a polyadenylation(pA) site. Due to size limitations, genes for use in rAAV vectorstypically do not include introns. rAAV vectors also include the 5′ ITRand 3′ ITR flanking the gene, which is referred to as a transgene. Thusa typical rAAV genome has the following structure, in order from 5′ to3′ on the sense strand: ITR—promoter—transgene ORF—pA—ITR, and in oneaspect of the present invention, the promoter includes a TRE and thetransgene ORF is that of a colorimetric, e.g., fluorescent protein.Methods of molecular cloning of rAAV transgene constructs, preparationof rAAV particles, and storage and use thereof are broadly-known andfurther technical details are unnecessary for one of ordinary skill inthe art to be able to construct useful rAAV vectors, and produce and userAAV particles as described herein. As indicated above, so long as thegene sequence is less than the packaging limit of rAAV or scAAV, it isuseful for production of a transduction particle as described herein.

AAV is but one of many robust and well-characterized viral vectorssuited for gene therapy, which also includes, without limitation,gammaretroviruses, lentiviruses, adenovirus, and herpes simplex virus.While AAV is likely preferred in many instances, other safe andeffective viral transducing particles can be developed based on thegenes described herein for use in the devices, systems and methodsdescribed herein.

Likewise, DNA, such as plasmid or other forms of DNA, optionallycombined with suitable transfection reagents, such as liposomes.

In aspects, compositions and methods are provided for delivery of a geneto excitable cells or secretory cells, such as nerve cells or neurons,e.g., to sensory neurons, or inhibitory neurons or nerve cells, thatencodes a protein comprising a ligand binding domain fused to afunctional or effector domain (transmembrane ion channel or ion poredomain). As described in further detail below, the protein may be amutated native (non-chimeric) protein, such as a mutated GIyR ora7-nicotinic acetylcholine receptor, or a chimeric protein, such as aprotein comprising a mutated α7-nicotinic acetylcholine receptor ligandbinding domain (LBD) and a GIyR transmembrane ion channel domain.

In all instances, ligand-gated ion channels and their respective LBDsand transmembrane domains, are broadly-known, and their nucleotide andamino acid sequences, including a large number of mutated sequences thatselectively bind exogenous ligands, and transcription control elements,such as promoters, are broadly-available in the literature and freedatabases and sources, such as GenBank, UniProt, Addgene, EPD(eukaryotic promoter database), see, U.S. Pat. No. 8,435,762 and U.S.Patent Application Publication No. 2018/0009862, etc., among many otherliterature and on-line sources, and do not need to be recited herein.Likewise, methods of preparing genes encoding such proteins and forexpressing such proteins in a tissue-specific manner is routine and neednot be described beyond what is provided herein. Nevertheless, exemplarynucleic acid constructs, nucleic acid and amino acid sequences,recombinant virus particles, and related methods and reagents areprovided herein for illustrative purposes, and as proof of concept.

Excitable cells or secretory cells include, for example and withoutlimitation, sensory nerves and neurons including, without limitation,CNS neurons, such as spinal cord cells, such as dorsal horn cells and/orbrain cells, including and without limitation a brainstem, hindbrain,midbrain or forebrain excitatory or inhibitory cell population.

The functional domain of the modified LGICs described herein is atransmembrane ion channel that can be cationic-selective oranion-selective. Cationic-selective (e.g., Na⁺—Ca²⁺-, and K⁺-selective)channels, such as that of the 5HT3 receptor (also, 5-HT3 receptor, or5-hydroxytryptamine type 3 receptor), and the α7-nicotinic acetylcholinereceptor, have an excitatory, depolarizing effect on a neuron, whileanion-selective (e.g., Cl⁻-selective) channels, such as that of theglycine receptor (e.g., GIyR) or GABA A receptor, have an inhibitory,hyperpolarizing effect on the neuron. In one aspect, for painmanagement, the ion channel is hyperpolarizing, that is, when active,that is, when bound to the agonist ligand, the ion channel decreases aneuron's membrane potential to values more negative (e.g., −90millivolts (mV)) than resting potential (e.g., −70 mV). In aspects, ahyperpolarizing ion channel is permeable to Cl⁻ or K⁺ ions, and therebydecreases neuron membrane potential when active. GIyR is permeable toCl⁻, and therefore, when active, transfers Cl⁻ ions into the neuron.Suitable ion channels include transmembrane domains of members of theCys-loop family of receptors. Non-limiting examples of suitablehyperpolarizing ion channels include: Glycine receptors; GABA receptors,such as GABA_(A) and GABA_(C) receptors; Glutamate-gated chloridereceptors.

In another aspect, for pain management, the ion channel is depolarizing,and the cell is an inhibitory neuron. For example, as indicated in Table1, NPPY, Gal, or PV promoters can be used to effectively targetinhibitory neurons in the dorsal horn.

In one aspect, the protein is a mutated ligand-gated ion channel, suchas GIyR, GABA_(A), α7-nicotinic acetylcholine receptor, or 5HT3receptor, having mutations in the protein causing enhanced selectivityof binding to exogenous ligands (ligands not naturally found in the cellin which the protein is expressed). In reference to binding of a ligand,by “selective to” it is meant either exclusive to or substantially orsufficiently exclusive binding to a ligand, such that the effect of theother ligand is insignificant or below a suitable or acceptablethreshold level to achieve a desired purpose. For example, an LGIChaving a mutated α7-nicotinic acetylcholine LBD can be selective to anexogenous small molecule compound, such as varenicline, such that theLGIC is activated by the exogenous small molecule compound, and not toany clinically-relevant or physiologically-relevant extent byacetylcholine. Selective binding to an exogenous ligand is, compared tobinding to an endogenous ligand, at least about 4-fold to at least about200-fold enhanced potency as an agonist to the LGIC, includingincrements there between (see, e.g., U.S. 2018/0009862). Optionally,though preferably in many instances, the LGIC exhibits reduced bindingto endogenous ligands (native ligands), such as in the case of theα7-nicotinic acetylcholine ligand binding domain, reduced, negligible,or no binding to acetylcholine, but enhanced binding to exogenousligands, such as, for example and without limitation, varenicline (e.g.,CHANTIX©).

Methods of modification or mutation of ligand-gated ion channelproteins, including production of chimeric proteins, able to bindselectively to exogenous ligands, and examples of such proteins arebroadly-known, and well within the skill of an ordinary artisan (see,e.g., U.S. Pat. No. 8,435,762; U.S. Patent Application Publication No.2018/0009862; U.S. Pat. No. 8,957,036, incorporated herein by referencein its entirety; International Patent Publication No. 2017/049252,incorporated herein by reference for its description of additionalmodified LGICs; Weir et al., Using an engineered glutamate-gatedchloride channel to silence sensory neurons and treat neuropathic painat the source (2017) Brain 140; 2570-2585; Kynagh, T., et al., AnImproved Ivermectin-activated Chloride Channel Receptor for InhibitingElectrical Activity in Defined Neuronal Populations (2010) J. Biol.Chem. 285(20):14890-14897; and Sternson, S. M, et al. Chemogenetic Toolsto Interrogate Brain Functions (2014) 37:387-407).

Chimeric ligand-gated ion channel proteins have ligand-binding domainsand transmembrane ion channel domains from different proteins, eitherfrom the same or different species. In aspects, the protein is achimeric protein comprising a LBD of a nicotinic acetylcholine receptor,such as a mutated ligand binding domain from the α7 nicotinicacetylcholine receptor. In one aspect, the chimeric protein is achimeric protein described in one of U.S. Pat. No. 8,435,762, or U.S.Patent Application Publication No. 2018/0009862, or International PatentPublication No. 2017/049252, each of which is incorporated herein byreference in its entirety for its technical disclosure of suitablechimeric proteins (modified LGICs) e.g., comprising a mutated α7nicotinic acetylcholine receptor binding domain (ligand binding domain,LBD) fused to an ion pore domain (IPD), e.g., from a 5HT3, a GIyR, or aGABA_(C) receptor, as well as for disclosure of other modified LGICs.Non-limiting examples of LGICs include, without limitation, Cys-loopreceptors, e.g., AChR such as a nAChR, e.g., a muscle-type nAChR or aneuronal-type nAChR, gamma-aminobutyric acid (GABA; such as GABA_(A) andGABA_(A-p) (also referred to as GABA_(C)) receptors, GIyR, GluClreceptors, and 5HT3 receptors), ionotropic glutamate receptors (iGluR;such as AMPA receptors, kainate receptors, NMDA receptors, and deltareceptors), ATP-gated channels (e.g., P2X), and phosphatidylinositol4,5-bisphosphate (PIP2)-gated channels, and the modified LGIC cancomprise sequences of any appropriate combination of LBD and ion channelof the preceding, modified be selective for an exogenous ligand. LBDsequences and transmembrane ion channel sequences may be obtained fromany species, such as human, mouse, rat, sheep, cow, pig, or simianspecies, so long as it is functional for the intended use, for example,in humans, when used to produce a modified LGIC. The LGIC may behomomeric, or multimeric, comprising one or more LGIC subunits that canbe the same or different.

In some aspects, a modified LGIC subunit described herein can include aLBD from a α7 nAChR. Exemplary amino acid sequences for a7-nicotinicacetylcholine receptor LBDs are provided in FIG. 2 (obtained from U.S.Patent Application Publication No. 2018/0009862). In various aspects, anα7-nicotinic acetylcholine receptor LBD is a homolog, orthologue, orparalog of the human an α7-nicotinic acetylcholine receptor LBD setforth in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. In variousaspects, an α7-nicotinic acetylcholine receptor LBD has at least 75percent sequence identity (e.g., at least 80%, at least 82%, at least85%, at least 88%, at least 90%, at least 93%, at least 95%, at least97% or at least 99% sequence identity) to SEQ ID NO: 18, SEQ ID NO: 19,or SEQ ID NO: 20. Exemplary amino acid sequences for chimeric LGICs areprovided in FIGS. 3A-3E (obtained from U.S. Patent ApplicationPublication No. 2018/0009862). In various aspects, a modified LGIC has asequence set forth in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, orSEQ ID NO: 24. In aspects, a modified LGIC has an amino acid sequencehaving at least 75 percent sequence identity (e.g., at least 80%, atleast 82%, at least 85%, at least 88%, at least 90%, at least 93%, atleast 95%, at least 97% or at least 99% sequence identity) to SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

For purposes of generating a genetic construct for expressing these, orany amino acids, the nucleotide sequence of the ORF used can have anysuitable sequence that can be translated to the desired amino acidsequence. Due to codon degeneracy, the nucleotide sequence can varygreatly, but codon usage may be the same or different from the naturalgene, and can be optimized for increased, or optimal, expression.

In calculating percent sequence identity, two sequences are aligned andthe number of identical matches of amino acid residues between the twosequences is determined. The number of identical matches is divided bythe length of the aligned region (i.e., the number of aligned amino acidresidues) and multiplied by 100 to arrive at a percent sequence identityvalue. The length of the aligned region can be a portion of one or bothsequences up to the full-length size of the shortest sequence. Alignmentof two or more sequences to determine percent sequence identity can beperformed using the computer program ClustalW2 (EMBL-EBI) and defaultparameters, which calculates the best match between a query and one ormore subject sequences, and aligns them.

FIGS. 3A-3E (excerpted from U.S. Patent Application Publication No.2018/0009862) show exemplary amino acid sequences of chimeric LGICs.Mutation of amino acid residue 77 (e.g., W77F or W77Y) resulted insensitivity to granisetron and tropisetron. Mutation of amino acidresidue 79 (e.g., Q79G) was most effective for several agonists.Mutations of amino acid residue 131 (e.g., L131G, L131A, L131M, orL131N) altered sensitivity to varenicline, tropisetron, granisetron, andACh. Potency was considerably enhanced when LBD mutations were combinedwith mutation at amino acid residue 298 in the GlyR or GABA_(C) IPD.Potency was also enhanced when a7nAChR LBD mutations were combined withmutation at amino acid residue G175 and P216.

Specific, and non-limiting examples of modified LBDs of LGICs (relativeto SEQ ID NOs: 18, 19, and 20) that change ligand binding specificityinclude, amino acid substitution at one or more of amino acid residues77, 79, 115, 131, 139, 141, 175, 210, 216, 217, and 219, including,without limitation: W77F, W77Y, W77M, Q79A, Q79G, Q79C, Q79D, Q79E,Q79H, Q79L, Q79P, Q79R, Q79S, Q79T, Q79W, Y1 15F, Q1 39A, Q139C, Q139D,Q139F, Q139G, Q139H, Q1391, Q139K, Q139L, Q139M, Q139N, Q139R, Q139S,Q139V, Q139W, Q139Y, L141A, L141F, L141P, L141G, L141H, L1411, L141 M,L141 N, L141Q, L141S, L141 V, L141W, G175K, G175A, G175F, G175H, G175M,G175R, G175S, G175V, P2161, and Y217F. In one aspect, the modified LBDshas the following combinations of point mutations: L131G and Q139L;L131G, Q139L, and Y217F; Q79G and L131G; L131G and Y217F; Q79S andL131G; or Q79S, L131G, and Q139L.

Point mutations that reduce binding to acetylcholine include Y1 15F,Q79R, Q139G, Q139V, Q1 39W, Q139Y, L141A, L141 Q, L141S, can be combinedwith any of the selectivity-inducing mutations as described herein, suchas Q79G and L141 F. These modifications can be combined with amino acidsubstitutions to the ion channel domain that can alter conductance (See,U.S. Pat. No. 8,435,762 and U.S. Patent Application Publication No.2018/0009862).

Various synthetic ligands, and the modified LGICs they bind to andactivate, e.g., LGICs including modified α7nAChR LBDs are provided in,see, U.S. Pat. No. 8,435,762 and U.S. Patent Application Publication No.2018/0009862 A1, as well in the priority application to the presentapplication, U.S. Provisional Patent Application No. 62/473,630 filedMar. 20, 2017, which is incorporated herein by reference in itsentirety.

The exogenous LGIC ligand can be a synthetic exogenous LGIC ligandselected from the group consisting of a quinuclidine, a tropane, a9-azabicyclo[3.3.1]nonane, a6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, and a1,4-diazabicyclo[3.2.2]nonane. When the synthetic exogenous LGIC ligandis a tropane, the tropane can be tropisetron, pseudo-tropisetron,nortropisetron, compound 723, compound 725, compound 737, or compound745. When the synthetic exogenous LGIC ligand is a quinuclidine, thequinuclidine can be PNU-282987, PHA-543613, compound 0456, compound0434, compound 0436, compound 0354, compound 0353, compound 0295,compound 0296, compound 0536, compound 0676, or compound 702. When thesynthetic exogenous LGIC ligand is a6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, theligand can be compound 765 or compound 770. When the synthetic exogenousLGIC ligand is a 1,4-diazabicyclo[3.2.2]nonane, the ligand can becompound 773 or compound 774 (U.S. Patent Application Publication No.2018/0009862 A1).

Certain LGIC agonists associate increased potency with specific LBDsubstitutions in modified LGICs. FIG. 4A (from U.S. Patent ApplicationPublication No. 2018/0009862 A1) shows LGIC agonists that exhibitenhanced potency with substituted a7nAChR LBD having a Q79G amino acidsubstitution. Additional LGIC agonists are shown in FIG. 4B (alsoexcerpted from U.S. Patent Application Publication No. 2018/0009862 A1).For example, α7^(Q79G)-GlyR^(A298G) can be controlled by tropisetron,and α7^(L131G,Q139L,Y217F)-GlyR can be controlled by varenicline.Further examples of modified LGICs include the ivermectin-responsiveglutamate-gated chloride channels described in Weir et al., which wereadministered in AAV9 transducing particles intrathecally to mouse spinalcords (Weir et al., (2017) Brain 140; 2570-2585, see, also, Kynagh, T.,et al., (2010) J. Biol. Chem. 285(20):14890-14897), and theivermectin-responsive GIyR LGIC described in U.S. Pat. No. 8,957,036.Additional examples of the large number of known, specific modifiedLGICs and their specific agonists is beyond the scope of thisdisclosure, and is unnecessary.

According to one aspect of the invention, a method of modulatingactivity of excitable cells or secretory cells, such as nerve cells orneurons, e.g., to sensory neurons, e.g, depolarization orhyperpolarization of a neuron, in a patient is provided, comprisingadministering to an excitable cell or a secretory cell, such as a nervecell or neuron of the patient a nucleic acid comprising a gene forexpressing a modified LGIC that selectively binds, and is gated by anexogenous ligand to the cell, and where expression of the gene is undertranscriptional control of a promoter specific to an excitable cell or asecretory cell, e.g., a sensory nerve cell or neuron, thereby expressingthe modified LGIC in the cell, and administering the exogenous ligand tothe patient, thereby activating the LGIC. The LGIC comprises an LBD anda transmembrane ion channel domain, for example and without limitation,according to any aspect described herein. Where hyperpolarization isdesired, the modified LGIC comprises a transmembrane ion channel domainthat is selective for Cl− or K+ ions, such as a GIyR or GABA_(A) orGABA_(C) ion channel domain. Where depolarization is desired, themodified LGIC comprises a transmembrane ion channel domain that isselective for Na⁺ or Ca²⁺ ions. In aspects, the LBD is an α7-nicotinicacetylcholine receptor LBD according to any aspect provided herein.

According to another aspect of the invention, a method of treating adisease or disorder associated with the nervous system in a patient. Inone aspect, the disease or disorder associated with the nervous systemis pain, such as chronic pain. In another aspect, the disease ordisorder associated with the nervous system is itch. The methodcomprises administering to an excitable cell or a secretory cell of thepatient, such as a nerve cell or neuron, e.g., to CNS cells, such asspinal cord cells or brain cells, such as a dorsal horn cell or asupraspinal cell, a nucleic acid comprising a gene for expressing amodified LGIC that selectively binds, and is gated by an exogenousligand to the cell, and where expression of the gene is undertranscriptional control of a promoter specific to a sensory neuron,thereby expressing the modified LGIC in the sensory neuron, andadministering the exogenous ligand to the patent, thereby activating theLGIC. The LGIC comprises an LBD and a hyperpolarizing transmembrane ionchannel domain introduced into an excitatory cell, or a depolarizing,excitatory transmembrane ion channel domain that is administered toinhibitory neurons. For example and without limitation, inhibitory ionchannel domains according to any aspect described herein, include atransmembrane ion channel domain that is selective for Cl⁻ or K⁺ ions,such as a GlyR or GABA_(A) or GABA_(C) ion channel domain. For exampleand without limitation, excitatory ion channel domains according to anyaspect described herein, include a transmembrane ion channel domain thatis selective for Na⁺ or Ca⁺² ions, such as a 5HT3 ion channel domain. Inaspects, the LBD is an α7-nicotinic acetylcholine receptor LBD accordingto any aspect provided herein. In aspects, the pain is localized chronicpain in a patient, such as from osteoarthritic conditions, surgicalimplants, wounds, scarring, fibrotic conditions, nerve damage, ordisease, visceral pain, muscle or deep tissue damage, spinal cordinjury, post herpetic neuralgia, metabolic disease such as diabetes,chemotherapeutic neuropathy, idiopathic peripheral neuropathy.

In methods of delivering nucleic acids encoding modified LGICs,according to any aspect described herein to a cell or to a patient, thenucleic acid may be delivered by any useful method, in any useful form,as is recognized by those of ordinary skill in the field of genetictherapies. The nucleic acid may be naked nucleic acid, such as aplasmid, deposited, for example and without limitation, by a colloidaldrug delivery method, such as liposomes, e.g., cationic liposomes, ornanoparticles, or as part of a recombinant viral genome, as arebroadly-known. In aspects, liposomes or nanoparticles comprising thenucleic acid are injected at a desired site, such as in or adjacent tospecific neuronal tissue. In other aspects, a recombinant viral particle(transducing particle), is delivered, for example, injected, at adesired site, such as in or adjacent to, or otherwise targeting specificneuronal tissue. In one aspect, the nucleic acid comprising a gene forexpressing the modified LGIC is an AAV (Adeno-Associated Virus) genome.In another aspect, the nucleic acid is injected into or adjacent to atissue containing the target excitable or secretory cell, such as a CNScell, e.g, a dorsal horn cell, a spinal cord cell, a brain cell, or asupraspinal cell. In another aspect, the nucleic acid is administeredsystemically, e.g., intravenously, and optionally in a delivery vehicle,such as an AAV particle that has a tropism to excitable or secretorycells, such as, for example and without limitation, AAV9, AAV-PHP.eB,AAV-PHP.S, or rAAV2-retro particles mentioned above, selective to brain,peripheral and/or spinal cord tissue. The nucleic acid may be injectedonce or more than once in order to establish sufficient expression ofthe modified LGIC in the target neuron cells. Suitable carriers orexcipients for use in delivery of the nucleic acid, as are known in therelated arts, may be included in the dosage form for delivery of thenucleic acid, such as in a liposomal or a recombinant viral transducingparticle.

An “excipient” is an inactive substance used as a carrier for the activeingredients of a medication. Although “inactive,” excipients mayfacilitate and aid in increasing the delivery, stability orbioavailability of an active ingredient in a drug product. Non-limitingexamples of useful excipients include: antiadherents, binders, rheologymodifiers, coatings, disintegrants, emulsifiers, oils, buffers, salts,acids, bases, fillers, diluents, solvents, flavors, colorants, glidants,lubricants, preservatives, antioxidants, sorbents, vitamins, sweeteners,etc., as are available in the pharmaceutical/compounding arts. Forexample, for delivery to a nerve cell by injection, a drug product mightcomprise the nucleic acid in the form of a viral particle, nanoparticle,or liposome, in a suitable solvent, such as saline or phosphate-bufferedsaline, and including a rheology modifier or thixotropic agent.

Suitable dosage forms for delivery of exogenous ligands as describedherein, include, without limitation, oral, percutaneous, or inhaleddosage forms, the formulation of which is within the skill of anordinary artisan (see, generally, Troy, DB, Editor, Remington: TheScience and Practice of Pharmacy, 21st Ed., Lippincott Williams &Wilkins (2005), pp. 745-849, for descriptions of various compositions,solutions, and dosage forms useful for administration of the describedcompounds, as well as methods of making such compositions, solutions,and dosage forms). The exogenous ligand is delivered in an amount, anddosage regimen, effective to achieve a desired therapeutic end-point,such as lessening pain. Determination of safe and effective amounts ofthe exogenous ligand is routine, and within the skill of an ordinaryartisan. Further, certain suitable exogenous ligands are approved foruse for other indications, such as varenicline, or tropisetron, and assuch suitable safe dosage ranges are already established in humans.

Example 1—Calretinin⁺ and CCK⁺ Neurons in the Dorsal Horn are Requiredfor Persistent Pain

Methods. Animals: All animals were kept on a standard 12:12 light/darkcycle in micro-isolator caging racks (Allentown Caging) with food andwater provided ad libitum. Mouse strains obtained from JacksonLaboratories include C57BI/6J (JAX #000664), Calr^(Cre) (JAX #010774)and CCK^(Cre) (JAX #012706). Adeno-associated viruses (AAV2/8) used inthese experiments: hSyn-Flex-rev-PSAM^(L141F)-GIyR-IRES-eGFP (7¹² vg/ml)and hSyn-Flex-rev-PSAM^(L)141F,Y115F-GyR-IRES-eGFP (1.7¹³ vg/ml) werecustom made by UNC Vector core based on plasmid material developed byScott Sternson and provided by Addgene. ThehSyn-Flex-rev-PSAM^(L)141F-GlyR-IRES-eGFP (7¹² vg/ml) was used in allexperiments except FIG. 8 (e,f). For these experiments,hSyn-Flex-rev-PSAM^(L)141F,Y115F-GyR-IRES-eGFP (1.7¹³ vg/ml) was used.Methods including: intra-spinal virus injections, paw withdrawalthreshold to von Frey filaments, to cotton swab, Hargreaves, pinprickand acute pain behaviors licking, guarding, flicking are all aspreviously described (Peirs, C. et al. Dorsal Horn Circuits forPersistent Mechanical Pain. Neuron 87, 797-812 (2015); Seal, R. P. etal. Injury-induced mechanical hypersensitivity requires C-low thresholdmechanoreceptors. Nature 462, 651-655, (2009)); For paw withdrawalthreshold to pressure: mice were lightly restrained by hand such thattheir rear legs were allowed to freely hang. Using a PressureApplication Measurement device (Ugo Basile) the hind paw was graspedbetween the experimenter's forefinger and thumb (with pressuretransducer on the thumb), and force was slowly applied to the paw untilthe mouse struggled or flicked its limb (paw withdrawal threshold, PWT).The final force in grams was recorded. Each mouse was tested on the leftand right paw for three trials with a ten-minute inter-trial intervalbetween applications, and the three results averaged for each paw.Injury Models including incision, complete Freund's adjuvant, sparednerve injury tibial and sural models, carrageenan, methylglyoxal andmulti-dose streptozotocin are as previously described (Peirs, C. et al.Dorsal Horn Circuits for Persistent Mechanical Pain. Neuron 87, 797-812(2015); Seal, R. P. et al. Injury-induced mechanical hypersensitivityrequires C-low threshold mechanoreceptors. Nature 462, 651-655, (2009);Decosterd, I. and Woolf, C. J. Spared nerve injury: an animal model ofpersistent peripheral neuropathic pain. Pain 87, 149-158 (2000);Bierhaus, A. et al. Methylglyoxal modification of Nav1.8 facilitatesnociceptive neuron firing and causes hyperalgesia in diabeticneuropathy. Nat Med 18, 926-933 (2012); Cavanaugh, D. J. et al. Distinctsubsets of unmyelinated primary sensory fibers mediate behavioralresponses to noxious thermal and mechanical stimuli. Proc Natl Acad SciU S A 106, 9075-9080, (2009)). Chemogenetic Activation of PSAM-GIyRReceptor: Behavior thresholds were performed as described above inAAV8-hSyn-DIO-PSAM-GIyR-IRES-GFP injected mice. PSEM^(89S) at 30 mg/kgwas injected intraperitoneally 15 minutes prior to testing.Immunohistochemistry was performed as previously described (Peirs, C. etal. Dorsal Horn Circuits for Persistent Mechanical Pain. Neuron 87,797-812 (2015); Seal, R. P. et al. Injury-induced mechanicalhypersensitivity requires C-low threshold mechanoreceptors. Nature 462,651-655 (2009)). α-bungarotoxin conjugated to Alexa-647 (1:1000;ThermoFisher B35450), α-bungarotoxin conjugated to Alexa-488 (1:1000;ThermoFisher B13422). Imaging: Spinal cord sections were imaged with aconfocal laser-scanning microscope (Nikon A1R) and Nikon Elementssoftware using 405-, 488-, 561- and 640 nm excitation laser light. Inorder to suppress emission crosstalk, the microscope was configured toperform all scanning in sequential mode. Z-series were scanned at 20×magnification with an oil immersion lens and a z-step of 5.0 μm.Quantification and Statistical Analysis: all data are reported asmean±SEM. For acute behaviors, a two-tailed Student's t-test was used.The effect of chemogenetic activation on persistent pain behavior wasanalyzed by one-way repeated measures ANOVA with Bonferroni's Post hoctest. Significance was considered p<0.05. (*p<0.05, **p<0.01,***p<0.001.) All quantitative analysis, graphs and statistical testswere performed on GraphPad Prism 7.0 (GraphPad).

Results. To determine the role of calretinin and cholecystokinin (CCK)excitatory dorsal horn neurons in persistent pain and baselinesomatosensory behavior, we used the designer ligand-gated anion channel,PSAM-GIyR (FIGS. 5, 8, and 10 ), which allowed us to acutely andreversibly inhibit these specific populations of neurons. Somatosensorybehavior was tested under chemogenetic control at baseline and after theinduction of inflammatory and neuropathic pain models (FIGS. 8 and 10 ).The inhibitory designer receptor, PSAM-GlyR¹⁶, is engineered such that amutated ligand-binding domain of the human α-7 nicotinic receptor isfused to the anion channel domain of the human glycine receptor (FIG.5(a), that is, FIG. 1 , panel (a)) (Magnus, C. J. et al. Chemical andgenetic engineering of selective ion channel-ligand interactions.Science 333, 1292-1296, (2011)). This mutated receptor binding domain nolonger recognizes physiological levels of acetylcholine, but insteadbinds the synthetic ligand PSEM^(89S) with high affinity. To selectivelytarget the CCK and Calretinin neurons in the dorsal horn, aCre-dependent adeno-associated virus (AAV) encoding PSAM-GIyR(AAV8-hSyn.FLEX.PSAM-GlyR.IRES.EGFP) was injected unilaterally into thedorsal horn of P21 CCK^(Cre) or Calretinin^(Cre) mice (FIG. 5(b)). Fourweeks later, receptor expression was examined by staining spinal cordslices with α-bungarotoxin conjugated to Alexa Fluor-647(α-BTX-Alexa647), which specifically recognizes the ligand bindingdomain of α-7 nicotinic acetylcholine receptors, including the ligandbinding domain of PSAM-GIyR (FIG. 5(b)). Expression of PSAM-GIyR waslargely limited to laminae III-IV (FIG. 5(b)) and did not overlap withPKCγ (PKCγ staining not shown). Specificity of α-BTX-Alexa647 stainingfor the exogenously expressed PSAM-GIyR was demonstrated by the lack ofstaining observed in the contralateral dorsal horn (FIG. 6 e ).

In Vitro and In Vivo Controls for the Specific Actions of PSEM89S onPSAM-GIyR

Prior to assessing the role of the neurons in somatosensory behavior,the ability of PSAM-GIyR to inhibit excitatory interneurons in thedorsal horn was tested using electrophysiological recordings in spinalcord slices. AAV8-Flex-PSAM-GIyR was injected intraspinally into thedorsal horn of P16 Tlx3^(Cre) mice. In this mouse line, the recombinaseis strictly expressed by excitatory neurons located throughout laminae1-Ill. Three weeks later, neuronal excitability was measured using patchclamp electrophysiology in spinal cord slices in the presence andabsence of PSEM^(89S) (FIG. 6 (a-d)). To identify PSAM-GlyR⁺ neurons, weidentified cells expressing green fluorescent protein (GFP), which isalso encoded by the PSAM-GIyR viral construct (FIG. 6(a,b)). Applicationof PSEM^(89S) (30 μM) to spinal cord slices significantly decreased themembrane potential (−7.3±3.4 mV, n=3 cells from 2 mice) and blockedaction potentials generated by current injection in GFP+(FIG. 6(c,d)),but not GFP⁻ neurons (data not shown). We also tested the effect of theligand PSEM^(89S) on pain behavior in mice lacking the receptor (FIG.6(f,h)). PSEM^(89S) injection had no effect on mechanical allodynia orheat hypersensitivity in the carrageenan, complete Freund's adjuvant orSNI pain model, suggesting the drug alone has no unintended behavioraleffects.

CCK+ Dorsal Horn Neurons are Required for Conveying Persistent Pain, butnot Baseline Somatosensory Behavior.

Three weeks after unilateral injection of AAV8-hSyn-Flex-PSAM-GlyR inthe dorsal horn of CCK^(Cre) mice, we tested the effect of inhibitingthe dorsal horn CCK neurons on baseline somatosensory behavior using vonFrey threshold, cotton swab assay, pinprick assay, pressure test andHargreaves assay (FIG. 7 ). We also tested motor behavior using rotarod(FIG. 7 ). All behaviors were normal and similar to the contralateralhindpaw (FIG. 7 ). We next tested the effect of inhibiting the dorsalhorn CCK neurons on persistent pain in the carrageenan model ofinflammatory pain. PSEM^(89S)injection significantly reversed punctateand dynamic mechanical allodynia as well as heat hypersensitivity (FIG.8(a)). Similarly, using the more chronic inflammatory model, completeFreund's adjuvant (CFA), PSEM^(89S) injection again significantlyreversed both punctate and dynamic allodynia (FIG. 8(b)) as well as heathypersensitivity. Injection of PSEM^(89S) (30 mg/kg, i.p.) into micelacking the receptor showed no effect on CFA-induced persistent pain(FIG. 6(g)). In the sural-SNI model of neuropathic pain,PSEM^(89S)injection significantly reversed both punctate and dynamicallodynia (FIG. 8(c)). Similarly, in the tibial-SNI model of neuropathicpain, PSEM^(89S) injection markedly reversed punctate allodynia (FIG.8(d)).

We have shown here that the CCK population is essential for thetransmission of carrageenan and CFA-induced heat hypersensitivity, butis dispensable for normal heat sensibility. Because heathypersensitivity also develops in diabetic neuropathic pain models, wetested whether the CCK neurons are also required in this type of pain.Indeed, acute inhibition of the CCK population markedly reversed theheat hypersensitivity induced by methylglyoxal (MG) treatment as well asthe heat hypersensitivity induced by the multi-dose STZ model ofdiabetic neuropathy (FIG. 8(e,f)). These data reveal for the first timean essential role for CCK excitatory dorsal horn neurons in conveyingpunctate and dynamic mechanical allodynia induced by inflammatory andneuropathic pain models. This population also has an important role inconveying heat hypersensitivity induced by inflammatory andpolyneuropathic pain models.

Calretinin neurons in lamina II of the dorsal horn are required forconveying mechanical allodynia induced by inflammatory injuries. Here weshow that calretinin expressing neurons in inner lamina II of the dorsalhorn are required for conveying mechanical allodynia induced byinflammatory injury. We unilaterally injected Cre-dependent AAV8PSAM-GIyR into the dorsal horn of Calr^(Cre) mice at P21 (FIGS. 5 and 10). Similar to what was observed when expressing the excitatory designerreceptor in this population, neurons expressing PSAM-GIyR wererestricted to inner lamina II (FIG. 5(b)) and co-localized withcalretinin, but not PKCγ (not shown). Baseline measures of mechanicaland heat sensitivity showed no change after injection of PSEM⁸⁹s ateither the ipsilateral or contralateral hindpaw. Motor behavior was alsonormal after PSEM^(89S) injection (FIGS. 9(a,b)). We next tested whetherthe calretinin neurons are required for conveying mechanical allodyniaor heat hypersensitivity in the carrageenan model of inflammatory pain.Injection of PSEM^(89S) 24 hours after carrageenan injectionsignificantly reversed punctate mechanical allodynia (FIG. 10(a)). Wealso tested whether the neurons are required for mechanical allodynia inthe CFA model. Measured 5 days after injection of the inflammatoryagent, injection of PSEM^(89S)resulted in a complete reversal ofpunctate mechanical allodynia at the ipsilateral hindpaw (FIG. 10(b)).To determine how generalizable the requirement for calretinin neurons isin conveying mechanical allodynia induced by inflammatory pain models,we tested the incision model of post-operative pain (FIG. 10(c)).Inhibition of the calretinin neurons also significantly reversedmechanical allodynia in this model. The data collected from a number ofinflammatory pain models show that dorsal horn neurons expressingcalretinin are required for conveying mechanical allodynia induced byinflammatory injuries.

Discussion Work shown here demonstrates that targeting of a designerligand-gated anion channel (in this case PSAM-GIyR) to neurons thatexpress CCK⁺ or calretinin⁺ in the dorsal horn markedly attenuatesmechanical allodynia and/or heat hypersensitivity caused by models ofinflammatory and neuropathic pain when the receptor is activated by thedesigner ligand (in this case PSEM^(89S)). The data also demonstratethat PSEM^(89S) and PSAM-GIyR mediated inhibition of the neurons doesnot affect baseline mechanical or thermal sensitivity. Finally, the datademonstrate that the ligand alone (i.e. in the absence of the receptor)does not affect mechanical or thermal sensitivity either before or afterinflammatory or neuropathic injury. Therefore, the data suggest thatinhibition of these neurons is sufficient to block persistent mechanicaland heat pain.

Example 2—Mechanical Allodynia Induced by Inflammatory and NeuropathicPain Models is Attenuated by Injection of Varenicline in Mice withmCCK-PSAM4 Delivered to the Dorsal Horn

As further proof of concept, a varenicline-responsive PSAM-GIyR receptormutant that was directly under the transcriptional control of a CCKpromoter and delivered to the dorsal horn was shown to markedlyattenuate mechanical allodynia in persistent pain models. The mousecholecystokinin (CCK) promoter with an added chimeric intron to driveexpression of α7^(L131G, Q139L, Y217F) GIyR (PSAM4) was packaged intoAAV2/8 and injected into the dorsal horn of 3-week-old wildtype C57BI/6male and female mice. Injection of AAV into the dorsal horn wasperformed as described previously (Peirs, C. et al. Dorsal Horn Circuitsfor Persistent Mechanical Pain. Neuron 87, 797-812 (2015)). See FIG. 11for a schematic of the plasmid used to produce these rAAV particles.Briefly, a 2.5 kb fragment of the mouse CCK promoter was subclonedupstream of the PSAM4 gene with the chimeric intron (135 bp) located inbetween. This construct expressed in AAV8 is sufficient to driveexpression in neurons in the deep dorsal horn.

Expression of the PSAM4 in the dorsal horn was examined byimmunostaining for α-BTX-Alexa647 (FIG. 12 ). α-BTX-Alexa647 stainingwas observed in cells in the deep dorsal horn below the PKCγ layer (PKCγstaining not shown). This expression pattern is similar to the patternfor CCK mRNA in the dorsal horn detected by in situ hybridization (AllenSpinal Cord Atlas) and by injecting AAV8 hSyn-Flex-PSAM-GIyR virus inthe dorsal horn of CCK^(Cre) mice (see FIG. 5(a)).

Varenicline has no effect on mechanical or heat sensitivity at baselineor after CFA or sural-SNI in the absence of PSAM4. Mechanical and heathypersensitivity was tested in adult wildtype male and female C57BI/6mice (not injected with mCCK-PSAM4 virus) before and afterintraperitoneal injection of (i.p) varenicline (0.1 milligram perkilogram (mpk) (FIG. 13 ). We also tested these parameters before and2-5 days after induction of the complete Freund's adjuvant (CFA)-modelof inflammatory pain and 7 days after induction of the sural version ofthe spared nerve injury (SNI) model of neuropathic pain (FIG. 13 ).Sensitivity to mechanical stimuli was tested by measuring the pawwithdrawal threshold to von Frey filaments (PWT) and the paw withdrawalresponse to a cotton swab (percentage of response) as describedpreviously (Peirs, C. et al. Dorsal Horn Circuits for PersistentMechanical Pain. Neuron 87, 797-812 (2015)). Heat sensitivity was testedby measuring the withdrawal latency to a radiant heat source using theHargreaves apparatus (PWL) as described previously (Peirs, C. et al.Dorsal Horn Circuits for Persistent Mechanical Pain. Neuron 87, 797-812(2015)). As shown in FIG. 13 , injection of varenicline (0.1 mpk, i.p)had no effect on baseline PWT, response frequency or PWL in uninjuredmice (measured 30-60 minutes after injection). Injection of vareniclinealso had no effect on mechanical or heat hypersensitivity 3 and 2 days,respectively, after induction of CFA or 7 days after induction ofsural-SNI models. PSAM4 ligands have no effect on baseline mechanical orheat sensitivity, but reverse mechanical allodynia induced by CFA andSNI in mice with targeted expression of PSAM4 in CCK+ neurons of thedorsal horn.

Mice were injected unilaterally in the dorsal horn with AAV8 mCCK-PSAM4virus and tested two weeks later. As shown in FIG. 14 , varenicline (0.3mpk, i.p.) did not alter baseline PWTs, response frequencies or PWLs.Thus, inhibition of the dorsal horn CCK+ neurons does not altermechanical or heat sensitivity. The ipsilateral plantar hindpaw of themice was injected with CFA and PWTs and response frequencies tested 3days later in the absence and presence of varenicline (0.3 mpk, i.p.).As shown in FIG. 15 , varenicline (0.3 mpk, i.p.) markedly reversed bothPWT and response frequency after CFA. As shown in FIG. 16 , varenicline(0.3 mpk, i.p.) markedly reversed the PWT when measured 7 days followingsural spared nerve injury. These results are similar to what we observedwith PSEM^(89S) and PSAM-GlyR in FIGS. 7 and 8 .

The following numbered clauses provide illustrative examples of aspectsof the invention:

-   -   1. A nucleic acid comprising a gene for expressing a modified        ligand-gated ion channel, comprising an open reading frame        encoding a modified ligand-gated ion channel under        transcriptional control of transcriptional control elements        governing cell-specific expression in CNS neurons, such as        dorsal horn neurons, spinal cord cells, or brain cells, or in        inhibitory neurons or nerve cells, such as a CCK promoter, a        Tac1 promoter, an NTS promoter, an NMU promoter, a Calb1        promoter, an SST promoter, a GRPR promoter, a parvalbumin        promoter, a Gal promoter, an NPY promoter, a PKCγ promoter or        Calb2 promoter, wherein the modified ligand-gated ion channel        comprises a modified ligand binding domain activatable by an        exogenous ligand, and optionally selective to the exogenous        ligand, and an ion pore domain.    -   2. The nucleic acid of clause 1, wherein transcription of the        modified ligand-gated ion channel is controlled at least in part        by a CCK promoter, an SST promoter, a GRPR promoter, a Tac1        promoter, an NTS promoter, an NMU promoter, a Calb1 promoter, a        parvalbumin promoter, a Gal promoter, an NPY promoter, a Calb2        promoter or a PKCγ promoter.    -   3. The nucleic acid of clause 1, wherein transcription of the        modified ligand-gated ion channel is controlled at least in part        by a CCK promoter, a Calb2 promoter, or a PKCγ promoter.    -   4. The nucleic acid of clause 1, wherein transcription of the        modified ligand-gated ion channel is controlled at least in part        by a CCK promoter.    -   5. The nucleic acid of any one of clauses 1-3, wherein the        transcription control element comprises a human promoter        sequence.    -   6. The nucleic acid of clause 5, wherein the transcription        control element is a promoter having at least 75 percent        sequence identity to a sequence set forth in SEQ ID NOs: 1-17.    -   7. The nucleic acid of any one of clauses 1-6, wherein the        modified ligand binding domain is a modified α7 nicotinic        acetylcholine ligand binding domain. 8. The nucleic acid of        clause 7, wherein the modified α7 nicotinic acetylcholine ligand        binding domain comprises a sequence having at least 75 percent        sequence identity to a sequence set forth in SEQ ID NO: 18, SEQ        ID NO: 19, or SEQ ID NO: 20.    -   9. The nucleic acid of clause 7, wherein the modified α7        nicotinic acetylcholine ligand binding domain comprises an amino        acid substitution at one or more of amino acid residues 77, 79,        115, 131, 139, 141, 175, 210, 216, 217, and 219.    -   10. The nucleic acid of clause 7, wherein the modified α7        nicotinic acetylcholine ligand binding domain comprises an amino        acid substitution at one or more of amino acid residues 77, 79,        115, 139, and 141, such as Q79A, Q79G, L141A, L141 F, L141 P,        W77F, W77Y, and W77M. 11. The nucleic acid of clause 7, wherein        the modified α7 nicotinic acetylcholine ligand binding domain        comprises:        -   a L131G amino acid substitution, a Q139L amino acid            substitution, and a Y217F amino acid substitution;        -   a W77F amino acid substitution, a Q79G amino acid            substitution, and a G175K amino acid substitution;        -   a Q79G amino acid substitution, a Y1.15F amino acid            substitution, and a G175K amino acid substitution;        -   a Y115F amino acid substitution and a G175K amino acid            substitution;        -   a Q79G amino acid substitution and a P2161 amino acid            substitution; or        -   a R27D amino acid substitution and/or a E41R amino acid            substitution.    -   12. The nucleic acid of clause 7, wherein the modified α7        nicotinic acetylcholine ligand binding domain comprises a L131 G        amino acid substitution, a Q139L amino acid substitution, and a        Y217F amino acid substitution.    -   13. The nucleic acid of clause 7, wherein the modified α7        nicotinic acetylcholine ligand binding domain has reduced        binding with endogenous acetylcholine (ACh) as compared to        unmodified α7-nAChR LBD.    -   14. The nucleic acid of clause one of clauses 1-13, wherein the        ion pore domain is anion-selective, or cation-selective, and        optionally is an ion pore domain of an ionotropic nicotinic        acetylcholine receptor, an ionotropic serotonin receptor, an        ionotropic glycine receptor, or an ionotropic GABA receptor.    -   15. The nucleic acid of any one of clauses 1-13, wherein the ion        pore domain is an ion pore domain from a serotonin 3 receptor        (5HT3) ion pore domain, a glycine receptor (GIyR) ion pore        domain, a gamma-aminobutyric acid (GABA) receptor ion pore        domain, or an α7 nicotinic acetylcholine receptor ion pore        domain.    -   16. The nucleic acid of clause 15, wherein the ion pore domain        is a GIyR ion pore domain comprising an amino acid substitution        at residue 298.    -   17. The nucleic acid of clause 16, wherein the GIyR ion pore        domain comprising an A298G amino acid substitution.    -   18. The nucleic acid of clause 1, wherein the exogenous ligand        is selected from the group consisting of a quinuclidine, a        tropane, a 9-azabicyclo[3.3.1]nonane, a        6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine,        and a 1,4-diazabicyclo[3.2.2]nonane.    -   19. The nucleic acid of any one of clauses 1-18, comprising a        sequence of a packageable viral genome comprising the gene for        expressing a modified ligand-gated ion channel.    -   20. The nucleic acid of clause 19, comprising Adeno-associated        virus ITR sequences flanking the gene for expressing a modified        ligand-gated ion channel, producing a sequence of a packageable        recombinant AAV genome or a self-complementary AAV genome        comprising the gene for expressing a modified ligand-gated ion        channel.    -   21. A method of modulating (increasing or decreasing) the        membrane potential of an excitable cell or a secretory cell,        comprising expressing in the cell a genetic construct comprising        a gene for expressing a modified ligand-gated ion channel,        comprising an open reading frame encoding a modified        ligand-gated ion channel under transcriptional control of        transcriptional control elements governing cell-specific        expression in CNS neurons, such as dorsal horn neurons, spinal        cord cells, or brain cells, or in inhibitory neurons or nerve        cells, such as a CCK promoter, a Tac1 promoter, an NTS promoter,        an NMU promoter, a Calb1 promoter, an SST promoter, a GRPR        promoter, a parvalbumin promoter, a Gal promoter, an NPY        promoter, a PKCγ promoter, or Calb2 promoter and a modified        ligand-gated ion channel comprising a modified ligand binding        domain activatable by an exogenous ligand, and optionally        selective to the exogenous ligand, and an ion pore domain, and        contacting the cell with an amount of the exogenous ligand        effective to activate the modified ligand gated ion channel        thereby modulating the membrane potential of the cell.    -   22. The method of clause 21, wherein transcription of the        modified ligand-gated ion channel is controlled at least in part        by a CCK promoter, an SST promoter, a GRPR promoter, a Tac1        promoter, an NTS promoter, an NMU promoter, a Calb1 promoter, a        parvalbumin promoter, a Gal promoter, an NPY promoter, Calb2        promoter or a PKCγ promoter.    -   23. The method of clause 21, wherein transcription of the        modified ligand-gated ion channel is controlled at least in part        by a CCK promoter, a Calb2 promoter, or a PKCγ promoter.    -   24. The method of clause 21, wherein transcription of the        modified ligand-gated ion channel is controlled at least in part        by a CCK promoter.    -   25. The method of any one of clauses 21-24, wherein the        transcription control element comprises a human promoter        sequence.    -   26. The method of clause 25, wherein the transcription control        element is a promoter having at least 75 percent sequence        identity to a sequence set forth in SEQ ID NOs: 1-17.    -   27. The method of any one of clauses 21-26, wherein the modified        ligand binding domain is a modified α7 nicotinic acetylcholine        ligand binding domain.    -   28. The method of clause 27, wherein the modified α7 nicotinic        acetylcholine ligand binding domain comprises a sequence having        at least 75 percent sequence identity to a sequence set forth in        SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.    -   29. The method of clause 27, wherein the modified α7 nicotinic        acetylcholine ligand binding domain comprises an amino acid        substitution at one or more of amino acid residues 77, 79, 115,        131, 139, 141, 175, 210, 216, 217, and 219.    -   30. The method of clause 27, wherein the modified α7 nicotinic        acetylcholine ligand binding domain comprises an amino acid        substitution at one or more of amino acid residues 77, 79, 115,        139, and 141, such as Q79A, Q79G, L141A, L141 F, L141 P, W77F,        W77Y, and W77M.    -   31. The method of clause 27, wherein the modified α7 nicotinic        acetylcholine ligand binding domain comprises:        -   a L131G amino acid substitution, a Q139L amino acid            substitution, and a Y217F amino acid substitution;        -   a W77F amino acid substitution, a Q79G amino acid            substitution, and a G175K amino acid substitution;        -   a Q79G amino acid substitution, a Y1.15F amino acid            substitution, and a G1.75K amino acid substitution;        -   a Y115F amino acid substitution and a G175K amino acid            substitution;        -   a Q79G amino acid substitution and a P2161 amino acid            substitution; or        -   a R27D amino acid substitution and/or a E41R amino acid            substitution.    -   32. The method of clause 27, wherein the modified α7 nicotinic        acetylcholine ligand binding domain comprises a L131 G amino        acid substitution, a Q139L amino acid substitution, and a Y217F        amino acid substitution, and, optionally, the exogenous ligand        is varenicline.    -   33. The method of clause 27, wherein the modified α7 nicotinic        acetylcholine ligand binding domain has reduced binding with        endogenous acetylcholine (ACh) as compared to unmodified        α7-nAChR LBD.    -   34. The method of clause one of clauses 21-33, wherein the ion        pore domain is an ion pore domain of an ionotropic nicotinic        acetylcholine receptor, an ionotropic serotonin receptor, an        ionotropic glycine receptor, or an ionotropic GABA receptor.    -   35. The method of any one of clauses 21-33, wherein the ion pore        domain is anion-selective, or cation-selective, and optionally        is an ion pore domain from a serotonin 3 receptor (5HT3) ion        pore domain, a glycine receptor (GlyR) ion pore domain, a        gamma-aminobutyric acid (GABA) receptor ion pore domain, or an        α7 nicotinic acetylcholine receptor ion pore domain.    -   36. The method of clause 35, wherein the ion pore domain is a        GIyR ion pore domain comprising an amino acid substitution at        residue 298.    -   37. The method of clause 36, wherein the GIyR ion pore domain        comprising an A298G amino acid substitution.    -   38. The method of clause 37, wherein the exogenous ligand-gated        ion channel ligand is selected from the group consisting of a        quinuclidine, a tropane, a 9-azabicyclo[3.3.1]nonane, a        6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine,        and a 1,4-diazabicyclo[3.2.2]nonane.    -   39. The method of any one of clauses 21-38, comprising a        sequence of a packageable viral genome comprising the gene for        expressing a modified ligand-gated ion channel.    -   40. A method of treating a disease or disorder associated with        the nervous system in a patient comprising: delivering a nucleic        acid according to any one of clauses 1-20 to the patient, and        administering the exogenous ligand to the patient in an amount        effective to activate the modified ligand gated ion channel in a        patient thereby treating the disease or disorder associated with        the nervous system in the patient.    -   41. The method of clause 40, wherein the disease or disorder        associated with the nervous system is itch.    -   42. The method of clause 40, wherein the disease or disorder        associated with the nervous system is chronic pain, and        transcription of the modified ligand-gated ion channel is        controlled at least in part by a CCK promoter, an SST promoter,        a GRPR promoter, a Tac1 promoter, an NTS promoter, an NMU        promoter, a Calb1 promoter, a parvalbumin promoter, a Gal        promoter, an NPY promoter, a Calb2 promoter or a PKCγ promoter.    -   43. The method of clause 25, wherein transcription of the        modified ligand-gated ion channel is controlled at least in part        by a CCK promoter, a calb2 promoter, or a PKCγ promoter.    -   44. The method of any one of clauses 40-43, wherein the nucleic        acid comprising the gene for expressing a modified ligand-gated        ion channel is delivered to the patient as a recombinant AAV        transducing particle.    -   45. The method of any one of clauses 40-44, wherein the        exogenous ligand is selected from the group consisting of a        quinuclidine, a tropane, a 9-azabicyclo[3.3.1]nonane, a        6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine,        and a 1,4-diazabicyclo[3.2.2]nonane.

While the present invention is described with reference to severaldistinct aspects or embodiments, those skilled in the art may makemodifications and alterations without departing from the scope andspirit. Accordingly, the above detailed description is intended to beillustrative rather than restrictive.

What is claimed is:
 1. A nucleic acid comprising a gene for expressing amodified ligand-gated ion channel, comprising an open reading frameencoding a modified ligand-gated ion channel under transcriptionalcontrol of a regulatory element governing cell-specific expression indorsal horn neurons, wherein the modified ligand-gated ion channelcomprises a modified ligand binding domain activatable by an exogenousligand, and an ion pore domain, wherein the modified ligand-gated ionchannel is a modified α7 nicotinic acetylcholine ligand binding domain,and wherein the ion pore domain is an ion pore domain of an ionotropicnicotinic acetylcholine receptor, an ionotropic serotonin receptor, anionotropic glycine receptor, an ionotropic GABA receptor, a serotonin 3receptor (5HT3) ion pore domain, a glycine receptor (GlyR) ion poredomain, a gamma-aminobutyric acid (GABA) receptor ion pore domain, or anα7 nicotinic acetylcholine receptor.
 2. The nucleic acid of claim 1,wherein the regulatory element is a promoter, a transcription responseelement, a repressor, and/or an enhancer.
 3. The nucleic acid of claim1, wherein the modified ligand binding domain is a modified α7 nicotinicacetylcholine ligand binding domain.
 4. The nucleic acid of claim 3,wherein the modified α7 nicotinic acetylcholine ligand binding domaincomprises a sequence having at least 75 percent sequence identity to asequence set forth in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:
 20. 5.The nucleic acid of claim 3, wherein the modified α7 nicotinicacetylcholine ligand binding domain comprises an amino acid substitutionat one or more of amino acid residues 77, 79, 115, 131, 139, 141, 175,210, 216, 217, or 219 of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20,such as Q79A, Q79G, L141A, L141 F, L141 P, W77F, W77Y, or W77M.
 6. Thenucleic acid of claim 3, wherein the modified α7 nicotinic acetylcholineligand binding domain comprises: a. a L131G amino acid substitution, aQ139L amino acid substitution, and a Y217F amino acid substitution; b. aW77F amino acid substitution, a Q79G amino acid substitution, and aG175K amino acid substitution; c. a Q79G amino acid substitution, aY115F amino acid substitution, and a G175K amino acid substitution; d. aY115F amino acid substitution and a G175K amino acid substitution; e. aQ79G amino acid substitution and a P2161 amino acid substitution; or f.a R27D amino acid substitution and/or a E41R amino acid substitution. 7.The nucleic acid of claim 3, wherein the modified α7 nicotinicacetylcholine ligand binding domain has reduced binding with endogenousacetylcholine (ACh) as compared to unmodified α7-nAChR LBD.
 8. Thenucleic acid of claim 1, wherein the exogenous ligand is selected fromthe group consisting of a quinuclidine, a tropane, a9-azabicyclo[3.3.1]nonane, a6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, and a1,4-diazabicyclo[3.2.2]nonane.
 9. The nucleic acid of claim 1,comprising a sequence of a packageable viral genome comprising the genefor expressing a modified ligand-gated ion channel.
 10. The nucleic acidof claim 9, comprising Adeno-associated virus ITR sequences flanking thegene for expressing a modified ligand-gated ion channel, producing asequence of a packageable recombinant AAV genome or a self-complementaryAAV genome comprising the gene for expressing a modified ligand-gatedion channel.
 11. A method of preparing a patient for a treatment forrelieving chronic pain, comprising: delivering to the patient a nucleicacid comprising an open reading frame encoding a modified ligand-gatedion channel under transcriptional control of a regulatory elementgoverning cell-specific expression in dorsal horn neurons, wherein themodified ligand-gated ion channel comprises a modified ligand bindingdomain activatable by an exogenous ligand, and an ion pore domain,wherein the modified ligand-gated ion channel is a modified α7 nicotinicacetylcholine ligand binding domain, and wherein the ion pore domain isan ion pore domain of an ionotropic nicotinic acetylcholine receptor, anionotropic serotonin receptor, an ionotropic glycine receptor, anionotropic GABA receptor, a serotonin 3 receptor (5HT3) ion pore domain,a glycine receptor (GlyR) ion pore domain, a gamma-aminobutyric acid(GABA) receptor ion pore domain, or an α7 nicotinic acetylcholinereceptor.
 12. The method of claim 11, wherein the nucleic acid isdelivered to the dorsal horn of the spinal cord of the patient.
 13. Themethod of claim 11, wherein the regulatory element is a promoter, atranscription response element, a repressor, and/or an enhancer.
 14. Themethod of claim 11, wherein the modified ligand binding domain is amodified α7 nicotinic acetylcholine ligand binding domain.
 15. Themethod of claim 14, wherein the modified α7 nicotinic acetylcholineligand binding domain comprises a sequence having at least 75 percentsequence identity to a sequence set forth in SEQ ID NO: 18, SEQ ID NO:19, or SEQ ID NO:
 20. 16. The method of claim 14, wherein the modifiedα7 nicotinic acetylcholine ligand binding domain comprises an amino acidsubstitution at one or more of amino acid residues 77, 79, 115, 131,139, 141, 175, 210, 216, 217, or 219 of SEQ ID NO: 18, SEQ ID NO: 19, orSEQ ID NO: 20, such as Q79A, Q79G, L141A, L141 F, L141 P, W77F, W77Y, orW77M.
 17. The method of claim 14, wherein the modified α7 nicotinicacetylcholine ligand binding domain comprises: a. a L131G amino acidsubstitution, a Q139L amino acid substitution, and a Y217F amino acidsubstitution; b. a W77F amino acid substitution, a Q79G amino acidsubstitution, and a G175K amino acid substitution; c. a Q79G amino acidsubstitution, a Y115F amino acid substitution, and a G175K amino acidsubstitution; d. a Y115F amino acid substitution and a G175K amino acidsubstitution; e. a Q79G amino acid substitution and a P2161 amino acidsubstitution; or f. a R27D amino acid substitution and/or a E41R aminoacid substitution.
 18. The method of claim 14, wherein the modified α7nicotinic acetylcholine ligand binding domain has reduced binding withendogenous acetylcholine (ACh) as compared to unmodified α7-nAChR LBD.19. The method of claim 11, further comprising administering theexogenous ligand to the patient in an amount effective to activate themodified ligand gated ion channel, thereby treating the chronic pain inthe patient.